Compositions and methods for topical delivery of pharmaceutical agents

ABSTRACT

Described herein are compositions comprising, and methods for using a composition comprising, Staphylococcal Exfoliative Toxin A (ETA) in an amount and duration sufficient to decrease the Stratum Corneum in a region of a subjects skin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims any and all benefits as provided by law,including the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 62/311,490 filed Mar. 22, 2016, which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2017, isnamed 030258-084692 SL.txt and is 5,139 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to transdermal delivery methods andformulations. Specifically, the invention relates to compositionscomprising, and methods of using compositions comprising StaphylococcalExfoliative Toxin A to decrease the Stratum Corneum of a subject.

BACKGROUND OF THE INVENTION

Transdermal delivery has potential advantages over other routes ofadministration. First-pass metabolism associated with oral delivery istypically reduced. Transdermal delivery is far less painful thaninjections. Therefore, this route of drug delivery should improvepatient compliance due to ease of applicability, improve efficacy byproviding localized release directly to affected sites and wheredesired, reduce toxicity by lowering systemic absorption.

In skin structures, the Epidermis serves to renew the epithelial layerand the top layer called the Stratum Corneum (S.C.) continuously. TheS.C. provides protection from the outer toxic environment and maintainsskin homeostasis to prevent water loss. Thus, the S.C. serves as theskin's primary barrier and consists of dead cells that are surrounded bya lipid extracellular matrix.

Effective topical delivery of large and often highly-chargedbiomolecules remains one of the most difficult challenges in dermatologydue to the skin's formidable S.C. permeability barrier. The barrierfunction of the S.C. prevents most hydrophilic and large molecularweight drugs (>300 Da) from penetrating intact skin. Efforts to modifyor bypass S.C., which include ballistic methods, laser, injection,ultrasound, iontophoresis and chemical depilation-induced anagen forhair follicles all require specialized equipment. These methods alsosuffer from challenges associated with toxicity and characteristicallyexhibit poor control over selective removal of S.C. and focal sites ofdelivery (e.g., hair follicles are targeted rather than contiguous skinor the epidermis is significantly disrupted). It would be desirable tomodify or remove S.C. in a controlled way in order to maintain epidermalviability, enhance permeability and improve methods of intradermal ortransdermal drug delivery.

SUMMARY OF THE INVENTION

Staphylococcal Exfoliative Toxin A (“ETA”) is a specific protease thatcleaves the extracellular portion of desmoglein-1 (Dsg1), anadherin-type cell-cell adhesion molecule found in stratified epithelialdesmosomes that mediates the adhesion of corneodesmosomes in the S.C. Ithas now been determined that the S.C. can be safely and effectivelytreated with ETA to increase the permeability of macromolecules,including pharmaceutical agents that are transdermally delivered.

In one aspect, the invention provides a method of reducing the StratumCorneum (S.C.) in a region of a subject's skin, said method comprisingtopically applying a composition comprising ETA in an amount andduration sufficient to decrease the S.C. in the region of the subject'sskin.

In one embodiment, the amount of ETA is between 5 μg/mL and 2000 μg/mL.

In another embodiment, the amount of ETA is between 100 μg/mL and 1500μg/mL.

In yet another embodiment, the amount of ETA is 1000 μg/mL.

In yet another embodiment, the composition comprising ETA is at a pH ofbetween about 6.0 and about 7.5.

In yet another embodiment, the composition comprising ETA is at a pH ofabout 6.5.

In yet another embodiment, the composition comprising ETA is at a pHbelow about 6.0.

In yet another embodiment, the composition comprising ETA is at a pHabove about 7.5.

In yet another embodiment, the composition comprising ETA is topicallyapplied at least once over a duration of at least 30 minutes, 1 hour or2 hours.

In yet another embodiment, the composition comprising ETA is topicallyapplied at least once over a duration of between 2 hours and 24 hours.

In yet another embodiment, the composition comprising ETA is topicallyapplied at least once over a duration of 12 hours, the compositioncomprising ETA is at a pH of 6.5, and the amount of ETA is 1000 μg/mL.

In yet another embodiment, the S.C. in the region is reduced bydesquamation.

In yet another embodiment, at least some of the S.C. in the regionregenerates after topically applying the composition comprising ETA.

In yet another embodiment, the S.C. in the region is regenerated after aduration of two weeks.

In yet another embodiment, the S.C. in the region of the subject's skinthat is reduced is between 1 cm² and 10 cm².

In yet another embodiment, acetone and/or a thioglycolate cream istopically applied to the region of the subject's skin prior to topicallyapplying a composition comprising ETA.

In yet another embodiment, the S.C. in the region of the subject's skinis reduced without damage to the epidermis or the dermis.

In another aspect, the invention provides a method of increasing anamount of at least one agent in the epidermis, or epidermis and dermis,within a region of a subject's skin, said method comprising topicallyapplying a composition comprising ETA to the region of the subject'sskin in an amount and duration sufficient to decrease the S.C. in theregion of the subject's skin, and applying an agent to the region of thesubject's skin, thereby increasing the amount of at least one agent inthe epidermis, or epidermis and dermis, within the region of thesubject's skin.

In one embodiment, the molecular weight of at least one agent is atleast 3000 Daltons, 5000 Daltons or 10,000 Daltons.

In yet another aspect, the invention provides a topical compositioncomprising an amount of ETA between 5 μg/mL and 2000 μg/mL at a pH ofbetween 6.0 and 7.5.

In one embodiment, the amount of ETA is 1000 μg/mL and the pH of thetopical composition is 6.5.

Other features and advantages of the invention will be apparent from theDetailed Description, and from the claims. Thus, other aspects of theinvention are described in the following disclosure and are within theambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying figures, incorporatedherein by reference.

FIG. 1 panels a-d depict a human skin explant from which the S.C. wasremoved following treatment with 50 μg/mL ETA at serial time points.Panel a depicts a control at 12 hours with the Stratum Corneum intact,panel b-c depicts times less than 12 hours and panel d depictssub-corneal cleavage and the complete removal of the S.C.

FIG. 2 panels a-d depict a sample of human skin explant treated indifferent buffers. Controls for a pH 7.4 HEPES buffer, panel a, and a pH6.5 PIPES buffer, panel c, demonstrate no desquamation. These compare toa skin explant in the pH 7.4 buffer for 4 hours, panel b, showing somedesquamation, and a skin explant in a pH 6.5 buffer for 4 hours, paneld, showing total desquamation.

FIG. 3 depicts the relationship of the concentration of ETA and the timelatency to desquamation with and without the effects of lowering pH inETA solution. All data were obtained from human skin specimens fromwhich the S.C. was removed following treatment with ETA at aconcentration range from 50 to 1000 μg/mL for designated hours ofincubation time. An increase in acidity to pH 6.5 led to a fasterreaction of ETA on human skin. In addition, the higher concentration ofETA reduced reaction time.

FIGS. 4A (panels a-f), 4B (panels a-d) and 4C (panels a-f) depictexperimental results of increased skin permeability to toxins on humanskin explants or xenograted human skin treated with ETA. The two headedarrows delineate the epidermis. FIG. 4A, depicts experimental resultsfor increased skin permeability to the cationic photo sensitizer EtNBS.FIG. 4B depicts increased skin permeability to Rhodamine-tagged Dextran,and FIG. 4C depicts increased permeability to FITC-human recombinantinsulin. The penetration of EtBNS was imaged at 2 hours (FIG. 4A, panela with toxin, panel d no toxin), 12 hours (FIG. 4A, panel b with toxin,panel e no toxin), and 24 hours (FIG. 4A, panel c with toxin, panel f notoxin). The progression of EtBNS is seen as the lighter shading, shownpenetrating past the epidermis in panel b and indicated by the encircledarea in panel c where the EtBNS has penetrated further. For Dextran,images were taken at 12 hours (FIG. 4B, panel a dye no toxin, panel cdye with toxin) and 24 hours (FIG. 4B, panel b dye no toxin, panel d dyewith toxin); showing penetration of the Rhodamine-tagged dextrin in theepidermis. The area encircled by the line in FIG. 4B panel d indicatesdeep penetration of the dye, which is seen as a pink coloration in thearea indicated. The toxin treatment allows the delivery of 3 kDa Dextranacross the skin. For FITC-insulin, images were taken at 6 hours (FIG.4C, panel a toxin, panel d no toxin), 12 hours (FIG. 4C, panel b toxin,panel e no toxin), and 24 hours (FIG. 4C, panel c toxin, panel f notoxin); showing penetration of the dye at 12 and 24 hours. The areacircled by the line in FIG. 4C, panel b indicates some dye penetration;which is seen as some green coloration in the area indicated. The areacircled by the line in FIG. 4C, panel c indicates deep penetration ofthe dye, which is seen as a bright green coloration in the areaindicated. The toxin treatment enables transcutaneous delivery ofInsulin-FITC (5000 Da). The green fluorescent dye penetrates within 6hours and extensively into the dermis after 24 hours.

FIGS. 5A-C depict an increase in skin permeability to severalpharmaceutical agents in ETA treated skin specimens measured using Franzcell diffusion apparatus assay. Each graph indicates the increase ofskin permeability to each dye in ETA-treated human skin compared tonon-treated control human skin. The pharmaceutical agents administeredare fluorescein (A) Sulforhodamine (B) Dextran, 3K Da (C). The squaredata markers indicate toxin treated human skin and the diamond datamarkers indicate control skin.

FIG. 6 panels a-d depict decreased desquamation time for the removal ofS.C. by ETA on human skin specimens following acetone pre-treatment.Samples either received no treatment (panel a), or treated with acetoneonly (panel c), ETA only (panel b), ETA and acetone (panel d). The arrowindicates complete separation of the S.C.

FIG. 7 panels a-d depict decreased desquamation time for the removal ofS.C. by ETA on the human skin specimen(s) following thioglycolate creampre-treatment. Samples either received no treatment (panel a), ETA only(panel b), treated with thioglycolate cream only (panel c), ETA andthioglycolate cream (panel d). The depictions are of live confocalimaging of skin. Images are focusing on the horizontal plane at the mostsuperficial levels. Lighter areas depict areas where DAPI dye hasentered the epidermis and staining on the Nuclei of the cells. When S.C.is removed, the DAPI dye directly enters epidermal cells, producingdiscrete cellular staining (swirls of staining with a fine dottedpattern of cellular uptake as seen in the panels). Natural folds in skinsurface produce “waves” of staining in these planar-focused live images.The waves of staining are seen in panel b, with about 20%-30% of thearea stained, and about 90%-95% of the area stained in panel d(“unstained” regions in panel d are skin folds). However when S.C. ispresent (panels a and c), DAPI cannot penetrate cells, and is shieldedfrom cellular uptake by the S.C., staining is seen to be less than about10% in panels a and c.

FIG. 8 panel a-f depict the improved desquamation time of ETA on S.C.removal following pre-treatment of samples with a combination of acetoneand thioglycolate cream. Immunofluorescence of DAPI signals usingconfocal imaging is depicted in panel a (control), panel b (ETA only),panel c (ETA with both pretreatments). Nuclei are seen only in panel c(which show up as small blue dots of stained discreet nuclei indicatingremoval of the Stratum Corneum), whereas only S.C. are seen in panels aand b (no discreet nuclei are seen). H&E histology images are depictedin panel d (control), panel (ETA only), and panel f (ETA with bothpretreatments).

FIGS. 9A (panel a and b), 9B, and 9C (panel a and b) depict thephenotype change of skin induced by Forskolin (FSK) application aftertoxin treatment. Forskolin has been shown to stimulate melanocytes toproduce melanin and darken murine skin. Forskolin cannot, however,penetrate intact human skin. FIGS. 9A and B shows selective induction ofpigmentation by topical Forskolin in the central zone (delineated by thesolid line) where ETA-desquamation was carried out, but not the adjacentintact human skin (FIG. 9A, panels a, b). FIG. 9B shows a comparison ofsamples treated with ETA and Forskolin (a), lower concentration offorskolin (b), and no treatment (c) after 7 days. FIG. 9C depictsSchmols staining (for eumelanin) of the desquamated Forskolin-treatedskin. Increased melanin production was induced by Forskolin in thedesquamated, Forskolin-treated area (panel b) as compared to little tono melanin in the control adjacent skin despite Forskolin applicationthroughout (panel a). Single headed arrows represent melanin deposition,double headed arrows depict S.C. Therefore, topical treatment withForskolin is seen to stimulate pigment synthesis only in the zone wherethe Stratum Corneum is removed. This indicates rescue of drugpenetration, and also indicates good health/function of the underlyingepidermal cells.

FIG. 10 panels a-h depict the effect of injected and topically appliedETA in a series of concentrations between 5 and 1000 ug/mL after 24hours. Damage caused by injection of ETA (panels a-d) was demonstratedcompared with the normal control skin and topically applied ETA control(panels e-h). Intradermal injection of ETA shows desquamation at 5 and50 μg/mL (panel a, b respectively), but also clefting at thedermal-epidermal junction (DEJ) at 500 and 1000 μg/mL (panels c, drespectively). Topical application of ETA shows no change at 5 μg/mL(panel e), but desquamation at 50, 500, and 1000 μg/mL (panels f-hrespectively).

FIG. 11 panels a-i depict DAPI staining on nuclei in an S.C. removedarea compared to control (intact S.C.), highlighting the recovery of theS.C. layer over time after the application of ETA on human xenograftedskin on mice. Days 1-3 (panel a, b) shows significant DAPI staining,which means that the whole surface of skin is desquamated. Day 4 (panelc) shows more staining than day 6 (panel d), which means more surfacearea of the skin is covered by new S.C., and day 6 preventing stainingof the nuclei. Day 7-9 and 10-11 (panels e, f) show even less nuclei.Day 12 (panel g) shows no nuclei and by day 15, only S.C. is shown, nonuclei are visible (panel h, i). The decrease in DAPI staining shows there-formation of intact Stratum Corneum which is complete by day 15.

FIGS. 12A (panel a-d) and 12B depict removal of S.C. by various methodsand possible damage. FIG. 12A panel a depicts controlled removal of S.C.using ETA with no epidermal or dermal injury. However, damage to theepidermis was shown when skin is treated with topical 30% salicylic acid(panel b), and 20% glycololic acid for 1-3 minutes (panel c) withepidermal damage and clefting of the dermal-epidermal junction (DEJ).Even more damage to the dermis and epidermis are seen in skin treated bydermabrasion (panel d). In FIG. 12A, single headed arrow points to theepidermis and double headed arrows show clefting between epidermis anddermis.

FIG. 12B shows skin treated with fractional laser showing dermal damageand heat injury (arrows).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control.

As used herein, a reduction in Stratum Corneum or “S.C.” refers to anamount of S.C. that is at least about 1-fold less (for example 1, 2, 3,4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold less) within aregion of a subject's skin than the amount of S.C. in the same region ofthe subject's skin prior to treatment according to the methods describedherein. A reduction in S.C. also means at least about 5% less (forexample 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99 or 100% less) within a region of a subject'sskin than the amount of S.C. in the same region of the subject's skinprior to treatment according to the methods described herein. Amountscan be measured according to methods known in the art.

As used herein “an increase in an amount of agent” refers to an amountof agent that is at least about 1-fold more (for example 1, 2, 3, 4, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) thanthe amount of agent in a subject without ETA treatment according to themethods described herein. “Increased” as it refers to an agent alsomeans at least about 5% more (for example 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%) thanthe amount of agent in the subject without ETA treatment according tothe methods described herein. Amounts can be measured according tomethods known in the art.

Staphylococcal Exfoliative Toxin A or “ETA” is a protease that cleavesthe extracellular portion of desmoglein-1 (Dsg1), an adherin-typecell-cell adhesion molecule found in stratified epithelial desmosomesthat mediates the adhesion of corneodesmosomes in the S.C.

Staphylococcus aureus, protein sequence can be found at GenBank:AAA17490.1 exfoliative toxin A; GenBank Accession, AAA17490, SEQ ID NO:01. The full Exfoliative toxin A; GenBank Accession, P09331 SEQ IDNO:02. Embodiments include variants having protein sequences found atGenBank accession KPE24689.1, KPE22683.1, KPE21505.1 and the like.

The “Stratum Corneum” or “S.C.” is the outermost layer of the epidermisand provides the protective barrier function of the skin. The S.C.consists of corneocytes and keratin surrounded by lipid regions. Ingeneral, the Stratum Corneum has a thickness between 10 and 40 μm.

As used herein, the term “desquamation” refers to the shedding of theoutermost membrane or layer of a tissue, such as the S.C. of the skin.

As used herein, the terms permanent “damage” or “injury” to theepidermis or the dermis refer to damage that has an irreversiblenegative clinical impact on the epidermis or the dermis, such asscaring, permanent depigmentation, hyperpigmentation, or sustainedabnormal Stratum Corneum production, such as hyperkeratosis.

As used herein, the terms “damage” or “injury” to the epidermis or thedermis refer to damage that has a reversible negative clinical impact onthe epidermis or the dermis (i.e., no permanent damage or injury) suchas reversible pigmentation changes, pealing or wound formation.

As used herein, the term “regenerate” refers to restoration and/orrepair of the S.C. through a process involving the turnover of deadkeratinocytes. The S.C. is formed by cornification, whereby livingkeratinocytes are transformed into non-living corneocytes, the principalcomponent of the S.C. The S.C., therefore, is regenerated as thecornification process is restored by keratinocytes in the epidermis.

A “subject” is a vertebrate, including any member of the class mammalia,including humans, domestic and farm animals, and zoo, sports or petanimals, such as mouse, rabbit, pig, sheep, goat, cattle and higherprimates.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (aswell as fractions thereof unless the context clearly dictatesotherwise).

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Other definitions appear in context throughout this disclosure.

Compositions and Methods of the Invention

The S.C. is viewed currently as a layer of protein-enriched corneocytesembedded in a lipid-enriched, intercellular matrix, the so-called bricksand mortar model. The “bricks” are corneocytes surrounded by a cornifiedcell envelope made up of proteins, mainly loricrin, filaggrin, andinvolucrin, and covalently bound to the hydroxyceramide molecules of alipid envelope. These “bricks” are embedded in a “mortar” of lipidbilayers. The so-called mortar contains a variety of intercellularlipids including, ceramides, free sterols and sterolesters,cholesterolsulfate, and free fatty acids. The S.C. continually renewsitself, and there is a steady state between the proliferation anddifferentiation process of keratinocytes and desquamation ofcorneocytes. Topical delivery of agents, e.g., pharmaceutical agents, ishindered by the S.C.

Three isoforms of exfoliatives toxins have been found in Staphylococcusaureus (e.g., ETA, ETB, ETD). They are glutamate specific serineproteases that specifically cleave a single peptide bond in theextracellular region of human desmoglein 1 (Dsg1, a desmosomal cadherintype cell-cell adhesion molecule). ETs are known to facilitate bacterialinvasion into mammalian skin. It has now been determined that ETA can besafely and effectively utilized to temporarily remove the S.C., therebyimproving topical delivery of agents to the epidermis, dermis orcombinations thereof without causing permanent damage to the epidermisor the dermis.

ETA possesses strong “lock and key” specificity for Dsg1. Because Dsg1is critical within only the S.C., ETA can cause very superficialdisruption without permanently damaging the underlying dermis orepidermis. Cutaneous application of ETA and/or its derivatives willallow for direct penetration of drugs, molecules, peptides, nucleicacids (e.g., DNAs, RNAs) or other topical agents to the epidermis anddermis, permitting localized delivery while minimizing systemic effects.This molecularly-targeted approach takes advantage of a naturallyoccurring enzyme to provide an unprecedented level of control forsuperficial desquamation.

ETA topical compositions of the invention are suitable for safe andeffective use within a range of concentrations. Accordingly, the amountof topical ETA in compositions of the invention is between 5 μg/mL and2000 μg/mL, for example, about 25 μg/mL (e.g., between 20 and 30 μg/mL),about 100 μg/mL (e.g., between 90 and 110 μg/mL), about 500 μg/mL (e.g.,between 490 and 510 μg/mL), about 1000 μg/mL (e.g., between 990 and 1010μg/mL), about 1500 μg/mL (e.g., between 1490 and 1510 μg/mL) or about2000 μg/mL (between 1990 and 2010 μg/mL). In specific embodiments, theconcentration of ETA is about 1000 μg/mL.

ETA topical compositions of the invention are also suitable for safe andeffective use within a pH range. Accordingly, the pH of topical ETA incompositions of the invention is between 6.0 and 7.5, or in specificembodiments, the pH of ETA is at a pH of about 6.5 (e.g., between 6.1and 6.9, between 6.2 and 6.8, between 6.3 and 6.7, between 6.4 and 6.6)

ETA can be produced by plasmid expression in E. coli followed by HPLCpurification to about 99% purity. ETA can optionally be lyophilizedprior to formulation.

Formulations of the compositions of the invention include those suitablefor topical administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, ointments, pastes, emulsions, sprays,creams, lotions, slurries, suspensions, foams, controlled releaseformulations, gels, patches, implants, water-oil bilayer compositions,water-oil-powder trilayer compositions, serums, powders, mousses,hydrogels, single-use applicators, and the like, suitable for topicaladministration to the patient.

The formulations described herein can include a dermatologicallyacceptable carrier (also referred to herein simply as a “carrier”) forthe composition. The phrase “dermatologically acceptable carrier”, asused herein, means that the carrier is suitable for topical applicationto the hair/scalp, has good aesthetic properties, is compatible with theETA in the composition, and will not cause any unreasonable safety ortoxicity concerns. A suitable carrier is selected to yield a desiredproduct form. Furthermore, the solubility or dispersibility of thecomponents may dictate the form and character of the carrier. In someembodiments, the carrier is present at a level of from about 50 wt % toabout 99 wt %, about 60 wt % to about 98 wt %, about 70 wt % to about 98wt %, or, alternatively, from about 80 wt % to about 95 wt %, by weightof the composition.

The carrier can be in a wide variety of forms. Non-limiting examplesinclude simple solutions (e.g., aqueous, organic solvent, or oil based),emulsions, and solid forms (e.g., gels, sticks, flowable solids, oramorphous materials). In certain embodiments, the dermatologicallyacceptable carrier is in the form of an emulsion. Emulsion may begenerally classified as having a continuous aqueous phase (e.g.,oil-in-water and water-in-oil-in-water) or a continuous oil phase (e.g.,water-in-oil and oil-in-water-in-oil). The oil phase of the presentinvention may comprise silicone oils, non-silicone oils such ashydrocarbon oils, esters, ethers, and the like, and mixtures thereof.

The aqueous phase comprises water, such as demineralized or distilledwater, for example. Other acceptable carriers that may be used in theaqueous carrier include, but are not limited to alcohol compounds, suchas ethanol. According to one embodiment, the composition comprisesalcohol, dipropylene glycol, and/or water.

Emulsions may further comprise an emulsifier. The composition maycomprise any suitable percentage of emulsifier to sufficiently emulsifythe carrier. Suitable weight ranges include from about 0.1 wt % to about10 wt % or about 0.2 wt % to about 5 wt % of an emulsifier, based on theweight of the composition. Emulsifiers may be nonionic, anionic, orcationic. Suitable emulsifiers are disclosed in, for example, U.S. Pat.Nos. 3,755,560 and 4,421,769, and McCutcheon's Detergents andEmulsifiers, North American Edition, pages 317-324 (1986), which areincorporated herein by reference in their entirety. Suitable emulsionsmay have a wide range of viscosities, depending on the desired productform. Non-limiting examples of emulsifiers include glyceryl stearate,polysorbate 60, and the PEG-6/PEG-32/glycerol stearate mixture soldunder the name of Trefose® by Gattefosse. An emulsion may contain afatty phase that may range from between about 5 wt % to about 80 wt %(e.g., between about 5 wt % to about 50 wt %) of the composition. Any ofthe emulsions described herein may contain one or more agents selectedfrom the group of oils, waxes, emulsifiers, and coemulsifiers. Examplesof oils, waxes, emulsifiers, and coemulsifiers used in formulations arewell-known in the art. An emulsifier and a coemulsifier may be presentin the composition in a proportion ranging from 0.3 wt % to about 30 wt% (e.g., between about 0.5 wt % to about 20 wt %) of the composition. Anemulsion may contain lipid vesicles.

ETA topical compositions of the invention can be formulated togetherwith other agents, including, but not limited to, agents for thetreatment of skin cancer, inflammatory diseases, benign neoplasms (e.g.hemangiomas), pigmentary disorders, diagnostic agents and cosmeticagents for skin rejuvenation and evening of skin tone. Such formulationscan be configured to control release of the ETA and the agents atdifferent rates, either sequentially or concomitantly.

The dosage schedule for this use, i.e., the dosing regimen, will dependupon a variety of factors, including the stage of the disease orcondition, the severity of the disease or condition, the general stateof the patient's health, the patient's physical status, age and thelike.

ETA topical compositions of the invention are topically applied at leastonce over a duration of at least 30 minutes, between 30 minutes and 2hours, between 2 hours and 24 hours, or in specific embodiments, theduration is about 12 hours (e.g., between 10 and 14 hours, between 11and 13 hours, between 11.5 and 12.5 hours).

Methods of the present invention reduce the S.C. in a region of asubject's skin following topical application of ETA in an amount andduration sufficient to decrease the S.C. in the region of the subject'sskin. Typically, the S.C. in the region of the subject's skin that isreduced is between 1 cm² and 10 cm². In other embodiments, even less ofthe S.C. is reduced, and the ETA application and resulting desquamationmay form a pattern (e.g., spots, rows ect).

At least some of the S.C. in the region regenerates after topicallyapplying the composition comprising ETA. Treatment of diseases withsevere hyperkeratosis (e.g., keratodermas, psoriasis) may be treated ina more aggressive manner with less post-treatment regeneration of theS.C. In the course of treatment of other disorders, the S.C. in theregion is fully regenerated (e.g., after a duration of two weeks).

Methods of the present invention are ideally suited for increasing anamount of at least one agent in the epidermis, or epidermis and dermis,within a region of a subject's skin. Accordingly, ETA can be topicallyapplied to the region of the subject's skin in an amount and durationsufficient to decrease the S.C. in the region of the subject's skin,followed by topical application of an agent to the same region of thesubject's skin. Without the barrier of the S.C., the agent or agents aretransferred to the epidermis, or epidermis and dermis. If desired,certain agents may be administered in dosages suitable to enter theblood stream. Accordingly, agents can be administered topically, asadjuvants, prior to, or concomitantly with, systemic administrationdirectly into the blood stream. Agents can also be administeredconcomitantly with ETA for ease of application.

Agents within a range of sizes can be successfully transferred into theepidermis, or epidermis and dermis, including agents of 3000 Daltons,5000 Daltons or 10,000 Daltons, and all sizes in between.

In some embodiments, ETA facilitates delivery of agents to treatdisorders where topical delivery is favored, yet is presently known tobe hindered by the S.C. For example, agents for therapy of skin cancer,inflammatory diseases, vaccination, benign neoplasms (e.g. hemangiomas),pigmentary disorders, psoriasis, actinic keratoses and diagnostic andcosmetic use can be topically administered following ETA-mediatedreduction of the S.C.

Other agents that can be administered together with, or following ETA,include but are not limited to, antimicrobial agents, antiseptic agentsand analgesic agents. Such agents can be administered sequentially(e.g., after ETA administration) or concomitantly, for example, in adual release dosage formulation.

Examples of agents for therapy of skin cancer include, but are notlimited to, dacarbazine, temozolomide, nab-paclitaxel, paclitaxel,carmustine, cisplatin, carboplatin, vinblastine, 5-fluorouracil,imiquimod, tamoxifen, thalidomide, temozolimid, angiostatin, endostatin,INFalpha-2b, peginterferon alpha-2b, ipilimumab, pembrolizumab,nivolumab, vemurafenib, dabrafenib, trametinib, imatinib and erivedge.

Examples of agents for therapy of actinic keratoses include, but are notlimited to, 5-fluorouracil, diclofenac, ingenol mebutate and imiquimod.

Examples of agents for therapy of psoriasis include, but are not limitedto, topical immunosuppressive agents, etanercept, psoralens,corticosteroids, calcipotriene and tazarotene.

Examples of agents for therapy of pigmentary disorders include, but arenot limited to, hydroquinone, psoralens, corticosteroids, alpha arbutin,kojic acid, alpha-hydroxy acids, (including glycolic acid), tazarotene,hydroquinone, lignin peroxidase (LIP) and triluma.

Examples of antimicrobial agents include, but are not limited to,penicillins and related drugs, carbapenems, cephalosporins and relateddrugs, erythromycin, aminoglycosides, bacitracin, gramicidin, mupirocin,chloramphenicol, thiamphenicol, fusidate sodium, lincomycin,clindamycin, macrolides, novobiocin, polymyxins, rifamycins,spectinomycin, tetracyclines, vanomycin, teicoplanin, streptogramins,anti-folate agents including sulfonamides, trimethoprim and itscombinations and pyrimethamine, synthetic antibacterials includingnitrofurans, methenamine mandelate and methenamine hippurate,nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol,pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin,ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin,glycopeptide, glyclyclycline, ketolides, oxazolidinone; imipenen,amikacin, netilmicin, fosfomycin, gentamycin, ceftriaxone, ziracin,linezolid, synercid, aztreonam, and metronidazole, epiroprim,sanfetrinem sodium, biapenem, dynemicin, cefluprenam, cefoselis,sanfetrinem celexetil, cefpirome, mersacidin, rifalazil, kosan,lenapenem, veneprim, sulopenem, ritipenam acoxyl, cyclothialidine,micacocidin a, carumonam, cefozopran and cefetamet pivoxil.

Examples of antiseptic agents include, but are not limited to, alcohols,quaternary ammonium compounds, boric acid, chlorhexidine andchlorhexidine derivatives, iodine, phenols, terpenes, bactericides,disinfectants including thimerosal, phenol, thymol, benzalkoniumchloride, benzethonium chloride, chlorhexidine, povidone iode,cetylpyridinium chloride, eugenol and trimethylammonium bromide.

Examples of anti-inflammatory agents include, but are not limited to,nonsteroidal antiinflammatory agents (NSAIDs), propionic acidderivatives such as ibuprofen and naproxen, acetic acid derivatives suchas indomethacin, enolic acid derivatives such as meloxicam,acetaminophen, methyl salicylate, monoglycol salicylate, aspirin,mefenamic acid, flufenamic acid, indomethacin, diclofenac, alclofenac,diclofenac sodium, ibuprofen, ketoprofen, naproxen, pranoprofen,fenoprofen, sulindac, fenclofenac, clidanac, flurbiprofen, fentiazac,bufexamac, piroxicam, phenylbutazone, oxyphenbutazone, clofezone,pentazocine, mepirizole, tiaramide hydrochloride, steroids such asclobetasol propionate, bethamethasone dipropionate, halbetasolproprionate, diflorasone diacetate, fluocinonide, halcinonide,amcinonide, desoximetasone, triamcinolone acetonide, mometasone furoate,fluticasone proprionate, betamethasone diproprionate, triamcinoloneacetonide, fluticasone propionate, desonide, fluocinolone acetonide,hydrocortisone vlaerate, prednicarbate, triamcinolone acetonide,fluocinolone acetonide, hydrocortisone and others known in the art,predonisolone, dexamethasone, fluocinolone acetonide, hydrocortisoneacetate, predonisolone acetate, methylpredonisolone, dexamethasoneacetate, betamethasone, betamethasone valerate, flumetasone,fluorometholone, beclomethasone diproprionate, fluocinonide, topicalcorticosteroids, and may be one of the lower potency corticosteroidssuch as hydrocortisone, hydrocortisone-21-monoesters (e.g.,hydrocortisone-21-acetate, hydrocortisone-21-butyrate,hydrocortisone-21-propionate, hydrocortisone-21-valerate, etc.),hydrocortisone-17,21-diesters (e.g., hydrocortisone-17,21-diacetate,hydrocortisone-17-acetate-21-butyrate, hydrocortisone-17,21-dibutyrate,etc.), alclometasone, dexamethasone, flumethasone, prednisolone, ormethylprednisolone, or may be a higher potency corticosteroid such asclobetasol propionate, betamethasone benzoate, betamethasonedipropionate, diflorasone diacetate, fluocinonide, mometasone furoate,triamcinolone acetonide.

Examples of analgesic agents include alfentanil, benzocaine,buprenorphine, butorphanol, butamben, capsaicin, clonidine, codeine,dibucaine, enkephalin, fentanyl, hydrocodone, hydromorphone,indomethacin, lidocaine, levorphanol, meperidine, methadone, morphine,nicomorphine, opium, oxybuprocaine, oxycodone, oxymorphone, pentazocine,pramoxine, proparacaine, propoxyphene, proxymetacaine, sufentanil,tetracaine and tramadol.

Examples of anesthetic agents include, but are not limited to, alcoholssuch as phenol; benzyl benzoate, calamine, chloroxylenol, dyclonine,ketamine, menthol, pramoxine, resorcinol, troclosan, procaine drugs suchas benzocaine, bupivacaine, chloroprocaine, cinchocaine, cocaine,dexivacaine, diamocaine, dibucaine, etidocaine, hexylcaine,levobupivacaine, lidocaine, mepivacaine, oxethazaine, prilocaine,procaine, proparacaine, propoxycaine, pyrrocaine, risocaine, rodocaine,ropivacaine, tetracaine, and derivatives, such as pharmaceuticallyacceptable salts and esters including bupivacaine HCl, chloroprocaineHCl, diamocaine cyclamate, dibucaine HCl, dyclonine HCl, etidocaine HCl,levobupivacaine HCl, lidocaine HCl, mepivacaine HCl, pramoxine HCl,prilocaine HCl, procaine HCl, proparacaine HCl, propoxycaine HCl,ropivacaine HCl, and tetracaine HCl.

Examples of vaccinations include, but are not limited to, vaccinationsfor influenza, hepatitis, diphtheria, tetanus, pertussis, streptococcus,human papillomavirus, tuberculous, measles, mumps, rubella, andrespiratory syncytial virus and any currently known vaccination that isintradermally administered.

Examples of agents for diagnostics include, but are not limited to,liposomal doxorubicin, cytarabine, and cisplatin, gold nanoparticles(e.g., for detection of DNA), fluorophore-loaded silica nanoparticles,quantum dots, carbon nanotubes, silicon nanowires, nanopores, potassiumhydroxide, giemsa, methylene blue and wright's stain.

The present invention is additionally described by way of the followingillustrative, non-limiting Examples that provide a better understandingof the present invention and of its many advantages.

Embodiments of the various aspects described herein can be illustratedby the following numbered paragraphs.

Paragraph 1. A method of reducing the Stratum Corneum (S.C.) in a regionof a subject's skin, said method comprising topically applying acomposition comprising Staphylococcal Exfoliative Toxin A (“ETA”) in anamount and duration sufficient to decrease the S.C. in the region of thesubject's skin.Paragraph 2. The method of paragraph 1, wherein the amount of ETA isbetween 5 μg/mL and 2000 μg/mL.Paragraph 3. The method of paragraph 1, wherein the amount of ETA isbetween 100 μg/mL and 1500 μg/mL.Paragraph 4. The method of paragraph 1, wherein the amount of ETA isabout 1000 μg/mL.Paragraph 5. The method of any one of paragraphs 1-4, wherein thecomposition comprising ETA is at a pH of between 6.0 and 7.5.Paragraph 6. The method of any one of paragraphs 1-4, wherein thecomposition comprising ETA is at a pH of about 6.5.Paragraph 7. The method of any one of paragraphs 1-6, wherein thecomposition comprising ETA is topically applied at least once over aduration of at least 30 minutes, 1 hour or 2 hours.Paragraph 8. The method of any one of paragraphs 1-6, wherein thecomposition comprising ETA is topically applied at least once over aduration of between 2 hours and 24 hours.Paragraph 9. The method of paragraph 1, wherein the compositioncomprising ETA is topically applied at least once over a duration ofabout 12 hours, the composition comprising ETA is at a pH of about 6.5,and the amount of ETA is about 1000 μg/mL.Paragraph 10. The method of any one of paragraphs 1-9, wherein the S.C.in the region is reduced by desquamation.Paragraph 11. The method of any one of paragraphs 1-10, wherein at leastsome of the S.C. in the region regenerates after topically applying thecomposition comprising ETA.Paragraph 12. The method of any one of paragraphs 1-11, wherein the S.C.in the region is regenerated after a duration of two weeks.Paragraph 13. The method of any one of paragraphs 1-12, wherein the S.C.in the region of the subject's skin that is reduced is between 1 cm² and10 cm².Paragraph 14. The method of any one of paragraphs 1-13, wherein acetoneand/or a thioglycolate cream is topically applied to the region of thesubject's skin prior to topically applying a composition comprising ETA.Paragraph 15. The method of any one of paragraphs 1-14, wherein the S.C.in the region of the subject's skin is reduced without damage to theepidermis or the dermis.Paragraph 16. A method of increasing an amount of at least one agent inthe epidermis, or epidermis and dermis, within a region of a subject'sskin, said method comprising topically applying a composition comprisingStaphylococcal Exfoliative Toxin A (“ETA”) to the region of thesubject's skin in an amount and duration sufficient to decrease the S.C.in the region of the subject's skin, and applying one or more agents tothe region of the subject's skin, thereby increasing the amount of atleast one of the agents in the epidermis, or epidermis and dermis,within the region of the subject's skin.Paragraph 17. The method of paragraph 16, wherein the amount of ETA isbetween 5 μg/mL and 2000 μg/mL.Paragraph 18. The method of any one of paragraphs 16-17, wherein theamount of ETA is between 100 μg/mL and 1500 μg/mL.Paragraph 19. The method of any one of paragraphs 16-18, wherein theamount of ETA is about 1000 μg/mL.Paragraph 20. The method of any one of paragraphs 16-19, wherein thecomposition comprising ETA is at a pH of between 6.0 and 7.5.Paragraph 21. The method of any one of paragraphs 16-20, wherein thecomposition comprising ETA is at a pH of about 6.5.Paragraph 22. The method of any one of paragraphs 16-21, wherein thecomposition comprising ETA is topically applied at least once over aduration of at least 30 minutes, 1 hour or 2 hours.Paragraph 23. The method of any one of paragraphs 16-21, wherein thecomposition comprising ETA is topically applied at least once over aduration of between 2 hours and 24 hours.Paragraph 24. The use of a topically applied composition comprisingStaphylococcal Exfoliative Toxin A (“ETA”) in an amount and durationsufficient to decrease the S.C. in the region of a subject's skin.Paragraph 25. The use of paragraph 24, wherein the amount of ETA isbetween 5 μg/mL and 2000 μg/mL.v26. The use of paragraph 24, wherein the amount of ETA is between 100μg/mL and 1500 μg/mL.Paragraph 27. The use of paragraph 24, wherein the amount of ETA isabout 1000 μg/mL.Paragraph 28. The use of any one of paragraphs 24-27, wherein thecomposition comprising ETA is at a pH of between 6.0 and 7.5.Paragraph 30. The use of any one of paragraphs 24-28, wherein thecomposition comprising ETA is at a pH of about 6.5.Paragraph 31. The use of any one of paragraphs 24-30, wherein thecomposition comprising ETA is topically applied at least once over aduration of at least 30 minutes, 1 hour or 2 hours.Paragraph 32. The use of any one of paragraphs 24-30, wherein thecomposition comprising ETA is topically applied at least once over aduration of between 2 hours and 24 hours.Paragraph 33. The use of paragraph 24, wherein the compositioncomprising ETA is topically applied at least once over a duration ofabout 12 hours, the composition comprising ETA is at a pH of about 6.5,and the amount of ETA is about 1000 μg/mL.Paragraph 34. The use of any one of paragraphs 24-33, wherein the S.C.in the region is reduced by desquamation.Paragraph 35. The use of any one of paragraphs 24-34, wherein at leastsome of the S.C. in the region regenerates after topically applying thecomposition comprising ETA.Paragraph 36. The use of any one of paragraphs 24-35, wherein the S.C.in the region is regenerated after a duration of two weeks.Paragraph 37. The use of any one of paragraphs 24-36, wherein the S.C.in the region of the subject's skin that is reduced is between 1 cm² and10 cm².Paragraph 38. The use of any one of paragraphs 24-37, wherein acetoneand/or a thioglycolate cream is topically applied to the region of thesubject's skin prior to topically applying a composition comprising ETA.Paragraph 39. The use of any one of paragraphs 24-38, wherein the S.C.in the region of the subject's skin is reduced without damage to theepidermis or the dermis.Paragraph 40. The use of a topically applied composition comprisingStaphylococcal Exfoliative Toxin A (“ETA”) and one or more topicallyapplied agents, wherein the ETA is applied in an amount and durationsufficient to decrease the S.C. in a region of a subjects skin, and theone or more agents is applied to the region of the subjects skin,thereby increasing the amount of at least one of the agent in theepidermis, or epidermis and dermis, within the region of the subjectsskin.Paragraph 41. The use of paragraph 40, wherein the amount of ETA isbetween 5 μg/mL and 2000 μg/mL.Paragraph 42. The use of paragraph 40, wherein the amount of ETA isbetween 100 μg/mL and 1500 μg/mL.Paragraph 43. The use of paragraph 40, wherein the amount of ETA isabout 1000 μg/mL.Paragraph 44. The use of any one of paragraphs 40-43, wherein thecomposition comprising ETA is at a pH of between 6.0 and 7.5.Paragraph 45. The use of any one of paragraphs 40-44, wherein thecomposition comprising ETA is at a pH of about 6.5.Paragraph 46. The use of any one of paragraphs 40-45, wherein thecomposition comprising ETA is topically applied at least once over aduration of at least 30 minutes, 1 hour or 2 hours.Paragraph 47. The use of any one of paragraphs 40-46, wherein thecomposition comprising ETA is topically applied at least once over aduration of between 2 hours and 24 hours.Paragraph 47. The use of ETA for the manufacture of a topically appliedmedicament to decrease the S.C. in skin.

EXAMPLES

The following Examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingExamples do not in any way limit the invention.

Example 1: Effective Concentration and Optimal pH for ETA Activity isDetermined

Patient compliance with a treatment regimen decreases as the difficultyof application and length of treatment increases. Concentrations of ETAwere evaluated to achieve optimum applications conditions.

ETA was mixed with 10 mM PIPES at pH 6.5, 10 mM HEPES at pH 7.4, or 10mM Tris at pH 8.0, at concentrations ranging from 25-1000 μg/mL. Eachcondition was also done separately as controls. These solutions wereapplied to human skin explants, obtained and prepared as follows. Humanskin specimens were obtained from consenting patients undergoingabdominoplasty. Under a sterile condition, the tissue was placed andwashed in a 70% ethanol solution and cold PBS. Fat layers were removedfrom the tissue, leaving a millimeter of dermis and an intact epidermis.The tissue was then cut into 10 mm×10 mm pieces and placed on gelscomposed of keratinocyte media supplemented with Human KeratinocyteGrowth Supplement purchased from Invitrogen, and mixed in a 1:1 ratiowith 2% agarose. The samples were stored at 37° C. in 5% CO2.

ETA solutions were incubated on top of the skin for 4 hours. Each buffercondition was also done separately without the presence of ETA ascontrols. After 4 hours incubation with ETA on human skin specimens,gentle scrubbing with wet autoclaved gauze resulted in desquamation ofhuman skin specimens. That is to say, Stratum Corneum layer was peeledoff mechanically with sterile gauze.

Human skin samples were fixed in 10% formaldehyde overnight and thenembedded in paraffin. Samples were cut using a microtome to a thicknessof 6 μM and baked on slides for 1 hour in a dry heat incubator at 58° C.Nuclei and eosinophillic structures were visualized by hematoxylin andeosin staining, respectively. The hematoxylin stains nuclei blue andeosins stains eosinophillic structures different shades of red.Histological images were taken with an inverted microscope.

At concentrations between 5 and 25 μg/mL, ETA was able to causedesquamation of the Stratum Corneum (“S.C.”) within 24 hours and withoutdamaging the rest of the epidermis or the dermis. At a concentration of50 μg/mL, ETA was able to remove the S.C. within 12 hours (FIG. 1),compared to the control, which had an intact S.C. after 12 hours with nosigns of apoptosis or cell death in the epidermal cells.

To shorten the incubation time of ETA in clinical applications, theenzyme's optimal pH was determined. A slightly acidic pH of 6.5 (FIG. 2)increased ETA's desquamation speed by completely removing the S.C. in 4hours compared to pH 7.4 (FIG. 2). Slightly basic buffer, 10 mM Trisbuffer pH 8.0, had no effect on the desquamation speed of ETA. Furtheracidic pH or too strong alkali treatment appeared to ruin skinstructure. Based on testing several parameters, it was determined that50 μg/mL of ETA in buffered pH 6.5 solutions provides optimal pHconditions for use in vivo.

With an increase in concentration of ETA, faster incubation time toremove S.C. was observed (FIG. 3). At a concentration of 1000 μg/mL in abuffered pH 6.5 solution, efficacy was achieved at about 3 hours ofincubation time.

In summary, an increase in the acidity to pH 6.5 resulted in a fasterreaction and a higher concentration of ETA (e.g., up to 1000 μg/mL)resulted in an improved reaction time (e.g., within 2 or 3 hours).

Example 2: Histological Analysis Shows Enhanced Permeability ofPharmaceutical Agents by ETA Treatment

Human skin samples were fixed in 10% formaldehyde overnight and thenembedded in paraffin. Samples were cut using a microtome to a thicknessof 6 μM and baked on slides for 1 hour in a dry heat incubator at 58° C.Nuclei and eosinophillic structures were visualized by hematoxylin andeosin staining, respectively. The hematoxylin stains nuclei blue andeosins stains eosinophillic structures different shades of red.Histological images were taken with an inverted microscope.

The skin was either subjected to no treatment or application of ETA at100 μg/mL. Subsequently, 50 μl of fluorescent dyes, includingfluorescein, sulforhodamine, dextran tagged with rhodamine and FITCtagged insulin, were applied topically on the surface of the skinexplant specimens.

To image the distribution of each dye in the human skin, human skinspecimens were collected at 6, 12, 18 and 24 hours. Ordinary skin withintact S.C. and desquamated skin using ETA (methods previouslydescribed) were used. Frozen sections of human skin specimens were cutusing a microtome to a thickness of 7 μM and DAPI was used for counterstaining. Skin imaging was carried out at room temperature using Nikonconfocal microscope equipped with NIS-Elements AR 3.10 software.

Removal of S.C. substantially increased skin permeability to EtNBS (400Da), Dextran (3 k Da), and human recombinant insulin (5K Da) on humanskin explants or xenograted human skin (FIG. 4). This indicates thatcontrolled removal of S.C. by ETA provides an effective way to increaseskin permeability to macromolecules that have previously been difficultto deliver topically.

Example 3: Franz Cell Apparatus Assay Shows Enhanced Delivery Efficacyof Pharmaceutical Agents by ETA Treatment

To quantify the delivery efficacy after ETA application, Franz cellassays were performed pertaining to fluoresceine, sulforhodamine B,dextran (3K), and human insulin (5K). Permeation experiments with Franzdiffusion cells were performed using human skin. The Franz diffusioncells were made of glass with a contact area of 1.35 cm², consisting ofa donor compartment and a receptor compartment. The skin was mountedbetween the cell compartments and 2 O-ring shaped glasses were used toposition the skin epidermis side-up and dermis side-down. The two cellcompartments were held together with a clamp. The receptor compartmenthad a volume of 4.3 ml and was filled with PBS-buffer. It was kept at37° C. on a heated plate. After 30 minutes of equilibration of the skinwith the receptor solution, 200 μl of the drug solution was applied inthe donor compartment with a pipet. The donor compartment was thencovered with parafilm to prevent evaporation of the solvent. Thereceptor solution was continuously stirred using a spinning bar magnetat 200 rpm. Receptor solution samples, 0.2 ml aliquots, were withdrawnthrough the sampling port of the receptor compartment at various timeintervals. The cells were refilled with a same amount of receptorsolution to keep the volume of receptor solution constant during theexperiment. The experiments were run for 24 hours. The fluorescentintensities collected from receptor compartments from normal anddesquamated skin were compared over time using a spectrophotometer.

The penetration of several fluorescent dyes was tested across fullthickness or split thickness (0.6 mm) of human skin for 24 hours andcompared with results from intact skin as a control. A substantialincrease of skin permeability to these molecules as measured by thespectrophotometer was observed in ETA treated skin specimens compared tocontrols (FIG. 5).

Next, the transcutaneous delivery of several dyes was further evaluatedby observation of each plane at 4 or 5 um deep under a Nikon confocalmicroscope. There were substantial increases in penetration offluorescent dyes after ETA application on skin explants.

Example 4: Varying Topical Conditions Allows for Effective Delivery ofETA in a Human Skin Explant Model

In order to decrease the incubation time of ETA, the followingconditions were additionally evaluated:

(1) Lipid Extraction (2) Protein Denaturation

Studies were carried out in a human skin explant model using surgicalabdominal skin specimens.

Lipid Extraction: The S.C. layer consists of lipids and therefore,acetone was selected as a pre-treatment to dissolve or at least disruptthe lipid structure of the S.C. A slight decrease in reaction time afterETA application was consistently observed following acetonepre-treatment (FIG. 6).

Protein Denaturation: The S.C. layer also contains keratin proteins.Keratins can be broken down by thioglycolate, which breaks down rigiddisulfide bonds. Samples were pre-treated with thioglycolate cream tobreak down the keratin layer of the S.C. As a result, the incubationtime of ETA was shortened (FIG. 7).

Combination Lipid Extraction and Protein Denaturation: With thepre-treatment of acetone and a thioglycolate cream, separation of S.C.started as early as 2 to 2.5 hours. Almost complete removal was seen at3 hours, even with a concentration of 50 μg/mL (at which an incubationof 4 to 8 hours is usually expected (FIG. 8).

Example 5: ETA Improves Drug Delivery and Induces Phenotypic Change inSkin Pigmentation

Forskolin, a cAMP inducer and potent MITF activator in the pigmentationcascade, was applied topically to human skin explants and observed dailyfor up to a week. Forskolin has been shown to increase skin pigmentationin mice but has not been able induce the same in human skin, potentiallydue in part to S.C. barrier function (Viros, A. et al. (2014) Nature.July 24; 511(7510):478-82; D'Orazio et al. (2006) Nature443(7109):340-4) but not human skin due to inability to penetrate.

As shown in FIGS. 9 (A) and (B), an increase in pigmentation byforskolin was noted in the area treated with topically applied ETAtoxin. No increase in pigmentation was demonstrated in the adjacentintact skin. This increase in pigmentation has not been demonstrated onhuman skin previously due to many hurdles in penetration, of which theS.C. is a major factor. Schmol's staining (FIG. 9C) demonstratedincreased melanin pigmentation in toxin treated area compared to thenon-desquamated adjacent skin as a control.

These experiments demonstrate the ability of ETA-mediated transdermaldelivery to induce biological function using pharmaceutical agents.

Example 6: Impaired Barrier Function is Repaired Following the Removalof S.C.

Damage caused by intra-dermal injection of ETA was compared to normalcontrol skin and topically applied ETA control. A very highconcentration of ETA (500 μg/mL) was intra-dermally injected into humanskin explants (prepared as described in Example 1) and observed after 24hours. At this concentration and method of application (i.e., IDinjection), a more wide spread exfoliation was noted and some cleavageplanes were found at the dermoepidermal junctions. Notably, in the caseof staphylococcal infection, the ETA reaches the S.C. through the bloodstream, traveling from the interior to the exterior side of the skin. Incontrast, methods of the invention topically apply ETA only to theoutside surface of skin. However, even at this high intra-dermalconcentration, no serious damage, including damage to the epidermis orunderlying dermis, was found (FIG. 10). With a topical ETA concentrationof 50 μg/mL for 24 hours, or even a higher concentration of ETA appliedfor a shorter period of time, a much more modest effect was observed(e.g., a topically-applied ETA at concentration of 1000 μg/mL stillrequires several hours to achieve desquamation of human skin (FIG. 3)).

Regarding healing kinetics, using a xenograft human skin in SCID micemodel, the healing of removed S.C. was completed within 2 weeks asdetermined by serial confocal observation of DAPI staining on xenograftskin. With confocal microscope and DAPI staining, the recovery of theS.C. can be observed, as described. DAPI is known to be a fluorescentstain that can pass through the cell membrane and bind strongly to A-Trich regions in DNA, thereby staining the nuclei of cells. With intactS.C., the signal from DAPI cannot be detected by confocal microscope dueto the refraction of light against the S.C. as well as the limitation ofthe depth of light penetration. On the other hand, desquamated skin canproduce DAPI signals within cells. DAPI can stain the nuclei ofepidermis as long as epidermis remains desquamated. Under inhalationanesthesia, xenografted mouse was taped down on the observatory platesand the xenografted skin was imaged using confocal microscopy. DAPIsignal was obtained at baseline and every other day after ETA-mediateddesquamation occurred. As shown in FIG. 11, less DAPI staining wasobserved over time, as S.C. was regenerated and repopulated thedesquamated area.

Subsequent desquamation demonstrates that the S.C. can be removed in acontrolled manner, as compared to conventional methods. Chemical peelingwith glycolic or salicylic acid, dermabrasion, and fractional ablativetechnology all lead to uncontrolled or unavoidable additional injury toepidermis and do not lead to desquamation of the S.C as depicted in FIG.12A. Fractional laser treatment lead to unavoidable heat damage on theside of the holds that each radiated beam made.

REFERENCES

All patents, patent applications and publications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent patent and publication was specifically andindividually indicated to be incorporated by reference.

SEQUENCES Exfoliative Toxin A [Staphylococcus aureus]ACCESSION AAA17490 (SEQ ID NO: 01)   1mnnskiiskv llslslftvg asafviqdel mqknhakaev saeeikkhee kwnkyygvna  61fnlpkelfsk vdekdrqkyp yntignvfvk gqtsatgvli gkntvltnrh iakfangdps 121kvsfrpsint ddngntetpy geyevkeilq epfgagvdla lirlkpdqng vslgdkispa 181kigtsndlkd gdkleligyp fdhkvnqmhr seielttlsr glryygftSvp gnsgsgifns 241ngelvgihss kvshldrehq inygvgigny (SEQ ID NO: 01)Full Exfoliative Toxin A; AltName: Full = Epidermolytic toxin A; Flags:Precursor. ACCESSION P09331 VERSION P09331.1 GI:119621 (SEQ ID NO: 02)  1 mnnskiiskv llslslftvg asafviqdel mqknhakaev saeeikkhee kwnkyygvna 61 fnlpkelfsk vdekdrqkyp yntignvfvk gqtsatgvli gkntvltnrh iakfangdps121 kvsfrpsint ddngntetpy geyevkeilq epfgagvdla lirlkpdqng vslgdkispa181 kigtsndlkd gdkleligyp fdhkvnqmhr seielttlsr glryygftvp gnsgsgifns241 ngelvgihss kvshldrehq inygvgigny vkriinekne (SEQ ID NO: 02)

1. A method of reducing the Stratum Corneum (S.C.) in a region of asubject's skin, said method comprising topically applying a compositioncomprising Staphylococcal Exfoliative Toxin A (“ETA”) in an amount andduration sufficient to decrease the S.C. in the region of the subject'sskin.
 2. The method of claim 1, wherein the amount of ETA is between 5μg/mL and 2000 μg/mL.
 3. The method of claim 1, wherein the amount ofETA is between 100 μg/mL and 1500 μg/mL.
 4. (canceled)
 5. The method ofclaim 1, wherein the composition comprising ETA is at a pH of between6.0 and 7.5.
 6. (canceled)
 7. The method of claim 1, wherein thecomposition comprising ETA is topically applied at least once over aduration of at least 30 minutes.
 8. The method of claim 1, wherein thecomposition comprising ETA is topically applied at least once over aduration of between 2 hours and 24 hours.
 9. (canceled)
 10. The methodof claim 1, wherein the S.C. in the region is reduced by desquamation.11-12. (canceled)
 13. The method of claim 1, wherein the S.C. in theregion of the subject's skin that is reduced is between 1 cm² and 10cm².
 14. The method of claim 1, wherein acetone and/or a thioglycolatecream is topically applied to the region of the subject's skin prior totopically applying a composition comprising ETA.
 15. (canceled)
 16. Amethod of increasing an amount of at least one agent in the epidermis,or epidermis and dermis, within a region of a subject's skin, saidmethod comprising topically applying a composition comprisingStaphylococcal Exfoliative Toxin A (“ETA”) to the region of thesubject's skin in an amount and duration sufficient to decrease the S.C.in the region of the subject's skin, and applying an agent to the regionof the subject's skin, thereby increasing the amount of at least oneagent in the epidermis, or epidermis and dermis, within the region ofthe subject's skin.
 17. The method of claim 16, wherein the amount ofETA is between 5 μg/mL and 2000 μg/mL. 18-19. (canceled)
 20. The methodof claim 16, wherein the composition comprising ETA is at a pH ofbetween 6.0 and 7.5.
 21. (canceled)
 22. The method of claim 16, whereinthe composition comprising ETA is topically applied at least once over aduration of at least 30 minutes, 1 hour or 2 hours.
 23. The method ofclaim 16, wherein the composition comprising ETA is topically applied atleast once over a duration of between 2 hours and 24 hours.
 24. Themethod of claim 16, wherein the composition comprising ETA is applied atleast once over a duration of 12 hours, the composition comprising ETAis at a pH of 6.5, and the amount of ETA is 1000 μg/mL. 25-26.(canceled)
 27. The method of claim 16, wherein the S.C. in the region ofthe subject's skin that is reduced is between 1 cm² and 10 cm².
 28. Themethod of claim 16, wherein acetone and/or a thioglycolate cream istopically applied to the region of the subject's skin prior to topicallyapplying a composition comprising ETA.
 29. (canceled)
 30. The method ofclaim 16, wherein the molecular weight of the at least one agent is 3000Daltons, 5000 Daltons or 10,000 Daltons.
 31. A topical compositioncomprising an amount of ETA between 5 μg/mL and 2000 μg/mL at a pH ofbetween 6.0 and 7.5.
 32. The topical composition of claim 31, whereinthe amount of ETA is 1000 μg/mL and the pH of the topical composition is6.5.